Formyl peptide receptors (FPRs) are a small group of G protein-coupled receptors that are known to be important in host defense and inflammation, and numerous studies have been carried out to identify small molecule ligands in order to characterize the structure and function of these receptors [1, 2]. The two receptors of the FPR family that are addressed here are FPR1, linked to antibacterial inflammation [3] and malignant glioma cell metastasis [4-6], and FPR2 (formally known as FPRL-1), linked to chronic inflammation in systemic amyloidosis, Alzheimer's disease, prion diseases and ischemia/reperpusion injury [7-12]. These two receptors were originally identified to be primarily expressed in myeloid cells, with varying distribution among myeloid cell subsets [1]. However, subsequent work has elucidated expression of functional receptors in a diversity of tissues including endothelial cells, hepatocytes, glial cells, astrocytes, platelets and olfactory neurons [1, 13].
There have been numerous studies to identify naturally occurring and synthetic ligands for each of these receptors (reviewed in [1, 11, 14]). Of particular relevance to the present work have been concerted efforts by a number of groups to develop progressively more potent small molecule agonists or antagonists for each receptor with therapeutic endpoints in mind. We previously reported the use of a fluorescent ligand competition assay and high-throughput flow cytometry to identify a series of novel small molecule ligands for FPR1 and FPR2 [15-17]. The most potent ligands identified were 3570-0208 and BB-V-115, with ligand binding inhibition constants (Ki) of 95 nM and 270 nM for FPR1 and FPR2, respectively [16]. Each was a selective antagonist of the intracellular Ca2+ response mediated by its target receptor with an IC50 value of 430 nM for FPR1 (3570-0208) and 940 nM for FPR2 (BB-V-115). Recently, two separate groups have identified more potent small molecule FPR1 antagonists with Ca2+ response IC50 values of 398 nM [18] and 4 nM [19]. Small molecule FPR1 agonists have been identified in a number of recent studies [2, 20-23], the most potent of which had a Ca2+ response EC50 value of 630 nM [21]. Potent FPR2 agonists have also been reported with Ca2+ response EC50 values in the 30-40 nM range [24, 25].
The search for novel ligands with high affinities to newly identified/poorly understood receptors remains one of the fundamental aims of biomedical research. Further understanding of a receptor's structure and function and biological relevance in certain diseases and disorders can be accelerated with the identification of high affinity ligands. Synthetic combinatorial methods have been in use for the last 20 years and have fundamentally advanced the ability to synthesize and screen large numbers of compounds. One of the earliest methods described, mixture-based libraries combined with positional scanning deconvolution is the approach that enables the most rapid and economical efficient acquisition of chemical and biological information [26-29]. The ability to identify specific functionalities responsible for driving the activity at each variable position of a chemical scaffold or pharmacophore is one of its strengths, and to this extent mixture-based libraries represent powerful tools that can be used for the identification of active individual compounds for a wide range of important biological targets.
Another advantage of mixture-based libraries resides in the very high densities of compounds that can be synthesized in highly dense regions of chemical space [30, 31] to quickly identify ‘activity cliffs’ defined as chemical compounds with high similar structure but unexpectedly very different biological activity [31, 32]. The diversity of the libraries synthesized by Torrey Pines Institute for Molecular Studies (TPIMS) has been characterized and described quantitatively by means of molecular scaffolds, molecular properties, and structural fingerprints. It has been shown that TPIMS libraries are unique in that there is partial overlap with the structural space of drugs, some libraries display scaffolds not present in other compound collections [31], and have increased molecular complexity as compared to compound libraries commonly used in high throughput screening (HTS) programs [30].
Extensive computational studies demonstrate that TPIMS libraries are excellent sources to identify selective compounds and expand the traditional relevant medicinal chemistry space typically covered by current commercial screening collections as well as the Molecular Library Small Molecular Repository (MLSMR) [30]. When compared to existing HTS programs, in which tens of thousands of individual compounds are screened against therapeutically important targets, millions of compounds formatted as mixtures can be examined using substantially less material and at much lower time/labor economics than if these same mixture-based diversities were made and screened as individual compounds. This unique combinatorial library approach can be applied to virtually any existing bioassay for the identification of novel ligands as has been reviewed [26, 27, 33].